Neisseria gonorrhoeae is a Gram-negative, strict human pathogen that causes the sexually transmitted disease, gonorrhea. The incidence of gonorrhea infections worldwide (estimated at over 60 million cases a year) and the ability of the bacteria to become resistant to most antibiotics used to treat infections makes it a global public health concern. Gonoccocal infections are usually initiated on mucosal surfaces, and require interaction of the bacteria with epithelial cells. One of the primary mediators of gonococcal attachment to mucosal surfaces is the Type IV pil (Tfp), which are polymers of pilin that extend from the bacteria and interact with receptors on epithelial cells. The goal of the proposed research is to define the regulatory mechanisms that control expression of Tfp in N. gonorrhoeae. The gene encoding the major pilin subunit (PilE) is subject to both phase and antigenic variation. We recently have identified a transcriptional regulator termed PssR (Pilus secretion system Represser Protein) that negatively regulates the pilus biosynthesis operon (pilMNOPQ) and the divergently transcribed penicillin-binding protein 1 (ponA) as well as a gene (rdgC) that encodes a protein involved in pilin antigenic variation. Moreover, our preliminary data indicate that PssR regulation of gene expression in N. gonorrhoeae may serve to modulate levels of bacterial susceptibility to innate host defense mechanisms, in particular the cationic antibacterial peptides that are postulated to be important in host resistance to infection. In order to better define the regulatory properties of PssR, we will determine its DNA-binding and transcriptional regulatory properties (Specific Aim 1). We will also determine the phenotypic effects of loss or over-expression of PssR relating to virulence and resistance to antimicrobials (Specific Aim 2). Since Tfp are critical for initiation of infection on mucosal surfaces of the genitourinary tract, studies on regulation of Tfp and/or other virulence determinants will advance our knowledge of how gonococci regulate virulence determinants and how their regulation contributes to pathogenesis.